The first step in reverse cholesterol transport is interaction of apolipoprotein A-l (apoA-l) with the ATP-binding cassette A1 (ABCA1) transporter, which is responsible for facilitating the transport and efflux of cholesterol from cells to form high density lipoprotein (HDL). Studies have indicated that ABCA1 preferentially transports low density lipoprotein (LDL)-derived cholesterol from late endosomes/lysosomes, a process that is regulated by the sterol-sensing Niemann-Pick C1 (NPC1) protein. It has been determined that other sterol-sensing proteins are negatively modulated by an insulin-inducible gene product called lnsig-1, however it remains unknown whether NPC1 is modulated in a similar fashion. The objective of this application is to determine the molecular basis for decreased apoA-l mediated cholesterol transport and efflux facilitated by ABCA1 when LDL-derived cholesterol-enriched human fibroblasts are incubated in the presence of increased insulin. The central hypotheses for this application is that increased insulin promotes lnsig-1 to negatively modulate NPC1, thereby causing inhibition of cholesterol transport and efflux from late endosomes/lysosomes facilitated by ABCA1. The rationale for conducting the proposed research is supported by studies indicating that lnsig-1 modulates the function of other sterol-sensing proteins by promoting retention at the endoplasmic reticulum, and that NPC1 transiently interacts with late endosomes/lysosomes and the endoplasmic reticulum. To test our central hypothesis and accomplish the objectives of this application, we propose the following specific aims. Specific Aim 1: Determine the expression of ABCA1, NPC1, and lnsig-1 resulting from the incubation of LDL-derived cholesterol-enriched human fibroblasts in the presence of low and high insulin. Specific Aim 2: Determine the cellular distribution and interaction of ABCA1, NPC1, and lnsig-1 resulting from the incubation of LDL-derived cholesterol-enriched human fibroblasts in the presence of low and high insulin. Specific Aim 3: Determine the transport of cholesterol into lipid rafts resulting from incubation of LDL-derived cholesterol-enriched human fibroblasts in the presence of low and high insulin. The proposed studies will have a significant impact on determining the mechanism of insulin resistance and/or hyperinsulinemia in causing premature ACVD and decreased HDL cholesterol with concomitant accumulation of cholesterol in cells of the artery wall. [unreadable] [unreadable] [unreadable]